Stabilization of Escherichia coli ribonuclease HI by strategic replacement of amino acid residues with those from the thermophilic counterpart.


Abstract

Thermus thermophilus ribonuclease H is exceptionally stable against thermal and guanidine hydrochloride denaturations as compared to Escherichia coli ribonuclease HI (Kanaya, S., and Itaya, M. (1992) J. Biol. Chem. 267, 10184-10192). The identity in the amino acid sequences of these enzymes is 52%. As an initial step to elucidate the stabilization mechanism of the thermophilic RNase H, we examined whether certain regions in its amino acid sequence confer the thermostability. A variety of mutant proteins of E. coli RNase HI were constructed and analyzed for protein stability. In these mutant proteins, amino acid sequences in loops or terminal regions were systematically replaced with the corresponding sequences from T. thermophilus RNase H. Of the nine regions examined, replacement of the amino acid sequence in each of four regions (R4-R7) resulted in an increase in protein stability. Simultaneous replacements of these amino acid sequences revealed that the effect of each replacement on protein stability is independent of each other and cumulative. Replacement of all four regions (R4-R7) gave the most stable mutant protein. The temperature of the midpoint of the transition in the thermal unfolding curve and the free energy change of unfolding in the absence of denaturant of this mutant protein were increased by 16.7 degrees C and 3.66 kcal/mol, respectively, as compared to those of E. coli RNase HI. These results suggest that individual local interactions contribute to the stability of thermophilic proteins in an independent manner, rather than in a cooperative manner. Study holds ProTherm entries: 734, 735, 736, 737, 738, 739, 740, 741, 2532, 2533, 2534, 2535, 2536, 2537, 2538, 13372, 13373, 13374, 13375, 14033, 14034, 14035, 14036, 14037, 14038 Extra Details: Activity determined at 30 degree C Escherichia coli ribonuclease HI; thermophilic; thermostability;,protein stability; thermal unfolding

Submission Details

ID: VucWcjSK4

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:15 p.m.

Version: 1

Publication Details
Kimura S;Nakamura H;Hashimoto T;Oobatake M;Kanaya S,J. Biol. Chem. (1992) Stabilization of Escherichia coli ribonuclease HI by strategic replacement of amino acid residues with those from the thermophilic counterpart. PMID:1328237
Additional Information

Number of data points 82
Proteins Ribonuclease HI ; Ribonuclease HI ; Ribonuclease HI
Unique complexes 6
Assays/Quantities/Protocols Experimental Assay: activity pH:5.5, ionic:-: -, temp:52.0 C ; Experimental Assay: ddG pH:5.5, temp:52.0 C ; Experimental Assay: activity buffers:Sodium acetate: 20 mM, pH:5.5, ionic:-: -, temp:52.0 C ; Experimental Assay: ddG pH:5.5, buffers:Sodium acetate: 20 mM, temp:52.0 C ; Experimental Assay: activity pH:3.0, ionic:-: -, temp:49.8 C ; Experimental Assay: ddG pH:3.0, temp:49.8 C ; Experimental Assay: activity pH:5.5, ionic:: , temp:25.0 C ; Experimental Assay: Cm buffers:glycine-HCl: 10 mM ; Experimental Assay: m buffers:glycine-HCl: 10 mM ; Experimental Assay: dG_H2O buffers:glycine-HCl: 10 mM ; Experimental Assay: activity pH:5.5, ionic:: ; Experimental Assay: Tm pH:5.5 ; Experimental Assay: dHvH pH:5.5 ; Experimental Assay: activity buffers:Sodium acetate: 20 mM, pH:5.5, ionic:: , temp:25.0 C ; Experimental Assay: Cm buffers:Sodium acetate: 20 mM ; Experimental Assay: m buffers:Sodium acetate: 20 mM ; Experimental Assay: dG_H2O buffers:Sodium acetate: 20 mM ; Experimental Assay: activity buffers:Sodium acetate: 20 mM, pH:5.5, ionic:: ; Experimental Assay: Tm pH:5.5, buffers:Sodium acetate: 20 mM ; Experimental Assay: dHvH pH:5.5, buffers:Sodium acetate: 20 mM ; Experimental Assay: activity pH:3.0, ionic:: ; Experimental Assay: Tm pH:3.0 ; Experimental Assay: dHvH pH:3.0 ; Derived Quantity: ddG_H2O buffers:glycine-HCl: 10 mM ; Derived Quantity: dTm pH:5.5 ; Derived Quantity: ddG_H2O buffers:Sodium acetate: 20 mM ; Derived Quantity: dTm pH:5.5, buffers:Sodium acetate: 20 mM ; Derived Quantity: dTm pH:3.0
Libraries Mutations for sequence MLKQVEIFTDGSCLGNPGPGGYGAILRYRGREKTFSAGYTRTTNNRMELMAAIVALEALKEHCEVILSTDSQYVRQGITQWIHNWKKRGWKTADKKPVKNVDLWQRLDAALGQHQIKWEWVKGHAGHPENERCDELARAAAMNPTLEDTGYQVEV
Sequence Assay Result Units